Kits for early detection of heart disease

ABSTRACT

The invention relates to methods, compositions, kits, and devices for detecting cardiac ischemia, hypoxia, or other causes of heart failure in a mammal by obtaining a test sample from a mammal, measuring a level of a non-polypeptidic cardiac marker in the test sample, and determining if the level of the cardiac marker measured in said test sample correlates with cardiac ischemia or hypoxia or another form of heart failure.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/084,069, filed May 22, 1998, by Sabbadini et al., and entitled“METHODS FOR EARLY DETECTION OF HEART DISEASE” now U.S. Pat. No.6,210,976, which in turn claims priority to the U.S. Provisional PatentApplication Serial No. 60/049,274, filed Jun. 10, 1997, by Sabbadini, etal., and entitled “DIAGNOSIS OF HEART DISEASES USING SPHINGOLIPIDS,”both of which are hereby incorporated by reference herein in theirentirety, including any drawings.

FIELD OF THE INVENTION

This invention relates generally to the area of diagnosis of heartdisease, and specifically relates to methods of diagnosis of heartfailure, cardiac ischemia, or hypoxia by detecting the level, e.g.,concentration, of a non-polypeptidic cardiac marker as an indicator ofheart damage, particularly chronic underlying coronary artery disease,and for monitoring of therapeutic regimes designed to alleviate cardiacischemia or hypoxia.

BACKGROUND OF THE INVENTION

Ischemic heart disease is the major form of heart failure. Heart failureaffects millions of people worldwide and is the leading cause of deathin the United States. The most common manifestation of cardiac ischemiais chest pain (angina pectoris) which can lead to heart attack (acutemyocardial infarction or AMI) and sudden death. In addition to those whoexhibit clinical symptoms of ischemic heart disease, many otherindividuals are at high risk of developing heart disease based onindicators such as hypertension conditions, high levels of serumcholesterol and/or family history.

Myocardial ischemic disorders occur when cardiac blood flow isrestricted (ischemia) and/or when the oxygen supply to heart muscle iscompromised (hypoxia) such that the heart's demand for oxygen is notmet. Atherosclerosis of the coronary artery is the most common cause ofischemia-associated symptoms such as angina pectoris. Ischemia andhypoxia can be transient and reversible, but can also lead toinfarction. During infarction, cardiac tissue is damaged and the heartcells become permeabilized, releasing a portion of their contents to thesurrounding milieu, including cardiac enzymes and other biochemicalmarkers. These cellular markers, such as creatine kinase (CK), lacticacid dehydrogenase (LDH) enzymatic activities and creatine kinase-MB(CKMB) and troponin (I and T) and myoglobin mass levels, are thendetectable in the serum.

Current diagnostic procedures generally assess the extent of cardiactissue damage after clinical signs have appeared. At that point,however, the disease may have progressed to an extent where AMI isimminent or has already occurred. Current methods of identifying andconfirming infarction require more time than is often available inemergency situations where rapid evaluation is critical for effectivepatient treatment and survival. Moreover, about 25% of AMI patientsdisplay atypical symptoms and many known tests result in falsenegatives, resulting in the unintentional discharge of about 5% ofpatients who have AMI (Mair J. et al., Clin. Chem. 41:1266-1272, 1995;Newby L. K. et al., Clin. Chem. 41:1263-1265, 1995). In an emergencymedical facility, electrocardiography (ECG) monitoring of suspected AMIpatients is the most rapid diagnostic method for detecting AMI, althoughit successfully detects only about half of AMI patients (Mair et al.,1995).

Electrocardiography and currently available diagnostic blood tests aregenerally not effective for early detection of myocardial ischemia thatprecedes the damage associated with AMI because the tests detectinfarction-associated tissue damage. They are not effective in earlydetection of chronic underlying coronary artery disease and theresulting myocardial ischemia that precedes the damage associated withAMI. Currently, the only diagnostic for chronic underlying coronaryartery disease is ECG monitoring during exercise stress (e.g., treadmillexercise) is generally used to confirm the clinical symptoms of angina.Such stress testing is usually given after the patient has experiencedsymptoms and sought treatment (e.g., at an emergency medical facility).Although stress testing is sometimes used to screen asymptomaticpatients, testing is costly, time-consuming and generally not amenableto routine screening of large numbers of patients. Furthermore, exercisestress test evaluations result in about 15% false negatives.

Diagnostics tests have been developed that use cardiac proteins todetermine whether or not the source of the patient's chest pain iscardiac and if so, whether the patient has suffered a myocardial infarctor is suffering from unstable angina (see, e.g., U.S. Pat. Nos.5,290,678, 5,604,105, and 5,710,008). These tests do not give an earlywarning for when myocardial infarct is forthcoming. Thus, anon-invasive, sensitive, and reliable point-of-care ‘bedside test’ isneeded for the early detection of cardiac ischemia, particularly forpeople at risk for heart disease.

In view of the need for rapid and reliable methods for detecting cardiacischemia in the absence of symptoms, particularly for screening those athigh risk of heart disease, the present invention is an early detectionassay for cardiac ischemia or hypoxia.

SUMMARY OF THE INVENTION

The present invention provides diagnostic methods for the earlydetection of heart disease (e.g., heart failure, cardiac ischemia, andcardiac hypoxia) in mammals, particularly humans, by monitoring serum orwhole blood levels of non-polypeptidic cardiac markers, e.g.,sphingosine and/or its metabolites. For instance, an early event in thecourse of cardiac ischemia (i.e., lack of blood supply to the heart) isan excess production by the heart. muscle of certain naturally occurringnon-polypeptidic compounds, or cardiac markers, such as, but not limitedto, sphingosine (SPH; D(+)-erythro-2-amino-4-trans-octadecene-1,3-diolor sphingenine), its isomers, and metabolites; ceramide (Cer,n-acylsphingosine), sphingosine-1-phosphate (S1P),sphingosylphosphorylcholine (SPC, lysosphingomyelin), andglycosphingolipids and lysophospholipids such as lysophosphatidic acid(LPA), and the metabolites of any of the foregoing. The presentinvention is based on the observation that SPH is increased in the serumand suggests that blood sphingolipid levels represent a new biochemicalmarker for cardiac ischemia.

Evidence indicates that the cardiac source of tumor necrosis factoralpha (TNFα) may be responsible for the characteristic increased serumsphingolipids resulting from cardiac ischemia. Accordingly, preferredembodiments of the invention provide that serum SPH levels, or levels ofother related lipids having a sphingosine backbone, be used incombination with levels of a secondary marker, e.g., serum TNFα, as anindex of ischemia. Of course, other non-polypeptidic cardiac markers canalso be used in conjunction with a secondary marker such as TNFα tocalculate such an index. This dual analyte measure is referred to asMyocardial Risk Factor (MRF).

Kits according to the invention provide cost-effective and rapid teststhat can be used to identify and predict, among other cardiacconditions, acute myocardial infarction (AMI) and to confirm that anginapectoris results from cardiac ischemia. In addition, the presentinvention can be used for simple screenings of early ischemic or hypoxicevents before symptoms are presented, e.g., in persons with high riskfor heart disease and for persons experiencing other forms of heartfailure, including myocarditis, the cardiomyopathies, and congestive andidopathic heart failure. Moreover, the methods and compositionsaccording to the invention can be used to monitor the effectiveness oftherapeutic interventions designed to relieve the ischemia and heartfailure.

Thus, in one aspect, the invention provides a method of detecting heartdisease characterized by cardiac ischeria or hypoxia in a mammalcomprising the steps of (a) measuring a level of a non-polypeptidiccardiac marker in the test sample from the mammal; and (b) determiningif the level of the cardiac marker measured in the test samplecorrelates with cardiac ischemia or hypoxia.

“Ischemia” means a condition where the cardiac muscle receivesinsufficient blood supply, whereas “hypoxia” means a condition where thecardiac muscle receives insufficient oxygen.

The term “mammal” refers to such organisms as mice, rats, rabbits,goats, horse, sheep, cattle, cats, dogs, pigs, more preferably monkeysand apes, and most preferably humans.

In preferred embodiments, the subject of the methods of the invention isa human, and the test sample used is preferably a body fluid. The bodyfluid is preferably selected from the group consisting of blood, urine,lymph, and saliva, although any other body fluid, such as serum, gastricjuices, and bile, may be used. Most preferably the body fluid is blood.

The term “non-polypeptidic cardiac marker” means a compound that is notconsidered to be a peptide by those skilled in the art, even though itmay contain a peptide bond or an amide bond, and is uniquely associatedwith the heart, such that the heart and cardiac functions are the sourceof the compound.

The non-polypeptidic cardiac marker is preferably a lipid and morepreferably a sphingolipid. A “lipid” means a substance that is insolublein water that can be extracted from cells by organic solvents of lowpolarity. Lipids include compounds such as terpenes, steroids, fats, andfatty acids. A “sphingolipid” means a compound that shares thesphingosine backbone containing an 18-carbon chain amino alcohol of thegeneral formula CH₃(CH₂)₁₄CH(OH)CH(NH₂)CH₂—R, where R may be any organicsubstituent. “Sphingosine” means the compound of formulaCH₃(CH₂)₁₄CH(OH)CH(NH₃ ⁺)CH₂OH, as shown in FIG. 1. The scope of theinvention also includes compounds where the carbon chain of thesphingolipid contains centers of unsaturation (i.e., double bonds ortriple bonds), or where hydroxide or the amine substituents are furthersubstituted with organic substituents. It is also understood“sphingolipid” refers to any isomer, e.g., threo-sphingosine,erythro-sphingosine, and L and D isomers of a sphingolipid, as well asany metabolite of any of the foregoing non-polypeptidic cardiac markers.

The non-polypeptidic cardiac marker is more preferably sphingosine orone of its metabolites. The metabolite is preferably selected from thegroup consisting of ceramide (Cer, n-acylsphingosine),sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), anddihydrosphingosine (DHSPH). The structures of these metabolites areshown in FIG. 1.

In preferred embodiments, the measuring step of the methods of theinvention comprises measuring the marker level by a method selected fromthe group consisting of chromatography, immunoassay, enzymatic assay,and spectroscopy, where the cardiac marker is directly or indirectlydetected. “Marker level” means the amount of the marker in the sample orin the mammal, and refers to units of concentration, mass, moles,volume, preferably concentration, or other measure indicating the amountof marker present in the sample.

The chromatographic method is preferably high performance liquidchromatography (HPLC) or gas chromatography (GC). The spectroscopicmethod is preferably selected from the group consisting of ultravioletspectroscopy ((UV or UV/V is spectroscopy), infrared spectroscopy (IR),and nuclear magnetic resonance spectroscopy (NMR).

The immunoassay preferably detects a non-polypeptidic cardiac markerselected from the group consisting of Cer, SPH, S1P, DHSPH, and SPC.Preferably, the immunoassay detects the non-polypeptidic cardiac markerin the test sample using anti-marker antibodies.

The term “antibody” refers to a monoclonal or polyclonal antibody orantibody fragment having specific binding affinity to a non-polypeptidiccardiac marker.

By “specific binding affinity” is meant that the antibody or antibodyfragment binds to target compounds with greater affinity than it bindsto other compounds under specified conditions. Antibodies or antibodyfragments having specific binding affinity to a compound may be used inmethods for detecting the presence and/or amount of the compound in asample by contacting the sample with the antibody or antibody fragmentunder conditions such that an immunocomplex forms and detecting thepresence and/or amount of the compound conjugated to the antibody orantibody fragment.

The term “polyclonal” refers to antibodies that are heterogeneouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

“Monoclonal antibodies” are substantially homogenous populations ofantibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art. See, for example, Kohler,et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110.

The term “antibody fragment” refers to a portion of an antibody, oftenthe hypervariable region and portions of the surrounding heavy and lightchains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the target compound. The term “antibody fragment”also includes single change antibodies.

In preferred embodiments, the determination step of the method ofinvention is a comparison between the concentration of the cardiacmarker and a predetermined value for the marker. In preferredembodiments, the predetermined value is indicative of a normal cardiaccondition. This predetermined value can be determined using the methodsof the present invention as described in Detailed Description of theInvention, below, and can be specific for a particular patient orgeneric for a given population. The predetermined value is preferablyobtained from a mammal in the same species and approximately the sameage as the mammal providing the test sample. In certain embodiments, thepredetermined value may have been established by prior measurement ofthe particular patient's marker levels when the patient was healthy.

In practicing the methods of the invention, the level.(e.g.,concentration) of the non-polypeptidic cardiac marker in the test sampleis preferably higher than a predetermined value for that marker, whichhigher level correlates with or indicates ischemia, hypoxia, or anotherform of heart failure. However, with certain non-polypeptidic cardiacmarkers, the level of the marker in the test sample may be lower thanthe predetermined value in order to indicate ischemia, hypoxia, oranother form of heart failure.

In a further aspect, the invention relates to a method of detectingheart failure (e.g., cardiac ischemia or hypoxia) in a mammal comprisingthe steps of (a) measuring a level of one or more non-polypeptidiccardiac markers in a test sample from the mammal; (b) measuring a levelof one or more secondary cardiac markers in the test sample; and ©determining if the level of the cardiac markers measured in the testsample correlates with cardiac ischemia or hypoxia The secondary cardiacmarker(s) is(are) preferably a pro-inflammatory cytokine such asinterleukin (IL-1, 2, or 6), interferon gamma (IFNγ), and particularlytumor necrosis factor alpha (TNFα). TNFα has been implicated in thepathophysiology of ischemia and hypoxia. As those in the art willappreciate, the instant methods and compositions may also includemeasurement of the levels of two (or) more non-polypeptidic cardiacmarkers, alone or in conjunction with one or more secondary cardiacmarkers. For purposes of this invention, a “secondary” cardiac marker isan intercellular or intracellular messenger which precipitates orcontributes to the underlying cause of heart failure. In otherembodiments of this aspect of the invention, the level of one or more“tertiary” cardiac markers can also be determined and used inconjunction with levels determined for the non-polypeptidic cardiacmarkers(s), or non-polypeptidic and secondary cardiac marker(s) tested.For purposes of this invention, a “tertiary” marker is one associatedwith disruption of cardiac cells, and generally relates to proteins,polypeptides, and nucleic acids released from ruptured or lyzed cardiaccells. Certain preferred examples of such markers include CK, LDH, CKMB,and troponin. Other preferred examples of such tertiary cardiac markersinclude nucleic acids specific for cardiac cells, particularly mRNA,expressed predominantly, and preferably only in cardiac cells.

In another aspect, the method of the invention concerns calculating amyocardial risk factor (MRF). As used herein, the MRF has a mathematicalrelation with the measured level, preferably concentration, of at leastone non-polypeptidic cardiac marker and the measured level, preferablyconcentration, of a second cardiac marker, e.g.,. TNFα. The mathematicalrelation is preferably a product of the measured level (e.g.,concentration) of at least one non-polypeptidic cardiac marker,preferably a sphingolipid, and the measured level (e.g., concentration)of the second marker, preferably TNFα. Of course, other mathematicalrelationships between different markers are also within the scope of theinvention. For example, such relationship may involve twonon-polypeptidic cardiac markers, a non-polypeptidic cardiac marker, asecondary cardiac marker, and a tertiary cardiac marker, or anon-polypeptidic cardiac marker and a tertiary marker.

In another aspect, the invention provides for a method of preventing orreducing the severity of a subsequent acute myocardial infarction (orother form of heart failure) by detecting cardiac ischemia or hypoxia,as described herein, and taking a preventive measure. The preventivemeasure is preferably selected from the group consisting of coronarybypass surgery, preventive angioplasty, and/or administeringtherapeutically effective amounts of one or more anticoagulants,thrombolytics, or other pharmaceutical products intended to alleviatethe ischemic or hypoxic condition.

Furthermore, the methods of the invention allow a health careprofessional to determine the prognosis of a patient following a cardiacprocedure by detecting cardiac ischemia or hypoxia. The cardiacprocedure is preferably selected from the group consisting of coronarybypass surgery, preventive angioplasty, and administering one or moreanticoagulant, although other cardiac procedures are also within thescope of the present invention.

In another aspect, the invention provides for kits for detecting heartfailure, such as may result from cardiac ischemia or hypoxia, in amammal. Preferably, such kits comprise a composition for detecting anabnormal level of at least one non-polypeptidic cardiac marker in a testsample obtained from a mammal. Preferably, the composition enablesmeasuring the abnormal level in a quantitative manner, althoughmeasuring the abnormal level can also be accomplished in asemi-quantitative manner (e.g., is the level above or below apre-determined threshold value). The composition may preferably comprisea substrate, which may preferably be an antibody which binds to anon-polypeptidic cardiac marker selected from the group consisting ofCer, SPH, S1P, DHSPH, and SPC. The composition may also include one ormore other substrates, e.g., an anti-TNFα antibody, to detect othercardiac-specific markers. The substrate may be affixed to a solidsupport for easy handling. Common forms of solid support include, butare not limited to, plates, tubes, and beads, all of which could be madeof glass or another suitable material, e.g., polystyrene, nylon,cellulose acetate, nitrocellulose, and other polymers. The solid supportcan be in the form of a dipstick, flow-through device, or other suitableconfiguration.

In a “quantitative” measurement, the step of measuring results in theproduction of a value which accurately shows the level of the cardiacmarker in the test sample. In a “semi-quantitative” measurement, thestep of measuring results in the indication of whether the level of thecardiac marker is within a particular range. Semi-quantitative methodsinclude, for example, but are not limited to, color indicators ordepiction of certain symbols, where each color or symbol represents aconcentration range.

Preferably, the level of the cardiac marker(s) detected in the practiceof this invention is(are) different than a standard or reference measurethat indicates a normal cardiac condition. More preferably, the level ofthe cardiac marker detected is greater than the standard measure.

In preferred embodiments, the level of the cardiac marker(s) measured inaccordance with the invention are detected using. a “non-invasive”method, i.e., one which does not require piercing the skin of thesubject mammal to obtain the test sample. Non-invasive methods include,but are not limited to, testing body fluids such as saliva, urine, andsweat, or using imaging techniques.

Preferably, the level of the cardiac marker is measured using a kit ofthe invention by a method selected from the group consisting ofchromatography, immunoassay, enzymatic assay, and spectroscopy, wherethe marker is directly or indirectly detected. The chromatographicmethod is preferably high performance liquid chromatography (HPLC) orgas chromatography (GC). The spectroscopic method is preferably selectedfrom the group consisting of ultraviolet spectroscopy, infraredspectroscopy, and nuclear magnetic resonance spectroscopy. With regardto the non-polypeptidic cardiac marker, the immunoassay preferablydetects Cer, SPH, S1P, DHSPH, or SPC.

In another aspect, the invention provides devices for detecting cardiacischemia or hypoxia in a mammal, where the device informs the user of anabnormal level of at least one non-polypeptidic cardiac marker in a testsample obtained from a mammal.

The informing step preferably includes the step of detecting saidcardiac marker, which, in turn, is preferably performed by anon-invasive procedure. The informing step also preferably comprises thestep of comparing the level of the marker with a predetermined value.Finally, the informing step preferably includes a step of alerting auser, who may or may not be the wearer of the device, as to the level ofthe marker. The device may display the level of the marker, sound analarm when the level of the maker surpasses a pre-determined threshold,or inform emergency personnel, such as police, ambulance, or firedepartment.

The mammal for whom the device is used is preferably a human. The devicepreferably tests a body fluid for the presence of a non-polypeptidiccardiac marker, which preferably is a sphingolipid, for example,sphingosine or a metabolite thereof. The sphingosine metabolite ispreferably selected from the group consisting of Cer, S1P, SPC, andDHSPH.

Yet another aspect of the invention concerns compositions for detectingan abnormal level (e.g., concentration) of at least one non-polypeptidiccardiac marker in a test sample (preferably a body fluid) obtained froma mammal, particularly a human. In certain embodiments, the level of thenon-polypeptidic marker is measured quantitatively; in otherembodiments, the measurement is semi-quantitative.

In preferred embodiments of this aspect, the composition comprises anantibody, anti-body fragment, or antigen binding domain of an antibody,that specifically binds a non-polypeptidic cardiac marker. Inembodiments employing an antibody, the antibody can be a polyclonal, andpreferably a monoclonal antibody. In certain embodiments, thenon-polypeptidic cardiac marker detected by the composition is a lipid,preferably a sphingolipid or a metabolite thereof, particularly Cer,SPH, S1P DHSPH, and SPC.

Compositions according to the invention may also comprise, in additionto a moiety capable of detecting a non-polypeptidic cardiac marker, asecond moiety capable of detecting a secondary cardiac marker (e.g.,TNFα, IL-1, IL-2, IL-6, and IFNγ), and/or a third moiety capable ofdetecting a tertiary cardiac marker (e.g., CK, CKMB, LPH, a troponin,and a nucleic acid, particularly a nucleic acid specific to cardiaccells). When the tertiary cardiac marker comprises a nucleic acid probesubstantially complementary to at least a sufficient portion of thenucleotide sequence of the nucleic acid so as to enable selectivehybridization between the probe and nucleic acid stringent conditions.

In preferred embodiments, the compositions of the invention furthercomprise a solid support to which the moiety detecting the cardiacmarker(s) is or can be attached. In certain embodiments, attachment ofthe detecting moiety, e.g. an antibody or nucleic acid probe, is via acovalent linkage with the solid support. In other embodiments,attachment may be via a non-covalent linkage, for example, betweenmembers of a high affinity binding pair. Many examples of high affinitybinding pairs are known in the art, and include biotin/avidin,ligand/receptor, and antigen/antibody pairs.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of sphingosine (SPH;D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol or sphingenine),sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC;lyso-sphingomyelin), ceramide (Cer, an n-acyl sphingosine) anddihydrosphingosine (DHSPH; sphinganine). All of these lipids share thesphingosine backbone containing a long-chain 18-C amino alcohol. Othersphingolipids include N,N-dimethyl-sphingosine, sphingomyelin(n-acylsphingosine-1-phosphocholine) and various glycosphingolipids(cerebrosides and gangliosides). Erythro, threo, D, L, and othersphingolipid isomers are also included within the scope of thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns methods and compositions for earlydiagnosis of ischemic heart disease or other forms of heart failure bydetecting levels non-polypeptidic cardiac markers, such as sphingosine(SPH) and/or its metabolites, alone or in conjunction with one or moreother cardiac markers in a test sample from a mammal. The invention isbased on the inventor's discovery that an early event in the course ofheart failure, for example, that caused by cardiac ischemia, is excessproduction by the heart muscle of certain non-polypeptidic cardiacmarkers, including certain lipids, among which are SPH and itsmetabolites, Cer, S1P, DHSPH, and SPC.

I. The Role of SPH in Myocardial Infarction

The chemical structures of Cer, SPH, S1P, and SPC are shown in FIG. 1.These sphingolipids all share the same chemical backbone ofCH₃(CH₂)₁₂CH═CHC(OH)CH(NH₂)CH₂— to which is attached either a hydroxyl,phosphate or phosphorylcholine moiety. As shown in FIG. 1, the aminogroup of the backbone can be positively charged or substituted. Althoughnot shown in FIG. 1, dihydrosphingosine (or sphinganine) is anothermetabolite of SPH known in the art (C. A. Grob, Record Chem. Progr.(Kresge-Hooker Sci. Lib.) 18:55-66, 1957; D. Shapiro, Chemistry ofSphingolipids (Hermann, Paris, 1969)). A variety of methods of detectingthese molecules in body fluids, e.g., blood or serum, can be used todetect actual or impending heart failure, such as that associated withmyocardial ischemic and hypoxic conditions. Based on results presentedherein, levels of SPH and/or its metabolites in body fluids provide anearly biochemical marker for cardiac ischemia or hypoxia

Sphingolipids (e.g., SPH, S1P, DHSPH, or SPC) can be extracted from theserum of patients with ischemic heart disease or controls withoutcardiac ischemic conditions and derivatized with a fluorescent marker(e.g.; o-pthalaldehyde, OPA) for chromatographic detection. Suchderivatized sphingolipids can then be detected and quantified by avariety of methodologies, including HPLC.

Although not wishing to be bound to a particular theory, data suggeststhat inflammatory cytokines, particularly TNFα, induce increasedproduction of SPH and its metabolites, either directly or indirectly.For example, it is believed that TNFα produces cardiac acidosis leadingto increased SMase activity and increased SPH production. The SPH thenacts on cardiac calcium channels, resulting in uncontrolled calciumrelease. The combined actions of TNFα and SPH also promote apoptosis,leading to increased release of intracellular SPH and its metabolitesinto the serum, and further leading to myocardial infarct. The inventorhas published data indicating that TNFα activates SPH production (Krownet al., J. Clin. Invest. 98:2854-2865, 1996), and that the resulting SPHand its metabolites has adverse effects on cardiac calcium channels(McDonough et al., Circ. Res. 75:981-989, 1994; Dettbarn et al., J. Mol.Cell. Cardiol., 26:229-242, 1994; Krown et al., FEBS Letters 376:24-30,1995; Sabbadini et al., J. Biol. Chem. 267:15475-15484, 1992; Webster etal., J. Mol. Cell. Cardio. 26:1273-1290, 1994) and cardiac cell death(Krown et al., J. Clin. Invest. 98:2854-2865, 1996).

Such cardiac hypoxia and ischemia result in a cycle whereby the acidicconditions of the ischemic heart stimulate excess SPH production which,in turn, inhibits the cell's ability to extrude protons. Increasedintracellular acidic conditions further stimulate SPH production in apositive feedback manner to further increase intracellular levels ofboth protons and SPH. The inventor believes that decreased intracellularpH has profound adverse effects on the cell's contractile machinery, andthat increased SPH levels cause the uncontrolled release of calcium fromthe sarcoplasmic reticulum membranes and the L-type calcium channel,thus preventing the cell from regulating its beat-to-beat contractilebehavior. Sphingolipid-mediated acidosis and calcium deregulationactivates apoptosis, leading to cell death and subsequent impairedcardiac function. SPH and its metabolites are useful as early indicatorsof heart failure because these compounds appear early in conditions suchas cardiac ischemia and hypoxia, before biochemical compounds associatedwith cardiac cell death are released.

This invention is based in part on the discovery that in ischemicpatients, the levels of serum sphingolipids are significantly higherthan those detected in non-ischemic controls. Based on the resultsobtained, levels of SPH that are diagnostic of heart failure associatedwith cardiac ischemia or hypoxia are generally greater than 100 pmol/mL.SPH levels diagnostic of cardiac ischemia or hypoxia are preferably in arange of about 200 pmol/mL to about 2,500 pmol/mL, more preferably in arange of about 300 pmol/mL to about 2,000 pmol/mL, and most preferablyin a range of about 400 pmol/mL to about 1,500 pmol/mL.

For the metabolites of SPH, high serum (or other body fluid) levels aresimilarly diagnostic of cardiac ischemia or hypoxia. For serum S1P,diagnostic levels are generally greater than 100 pmol/mL. In serum, S1Plevels diagnostic of cardiac ischemia or hypoxia are preferably in arange of about 200 pmol/mL to about 2,500 pmol/mL, more preferably in arange of about 300 pmol/mL to about 2,000 pmol/mL, and most preferablyin a range of about 400 pmol/mL to about 1,500 pmol/mL. For SPC,diagnostic levels in serum are generally greater than 100 pmol/mL. SPClevels diagnostic of cardiac ischemia or hypoxia are preferably in arange of about 200 pmol/mL to about 2,500 pmol/mL, more preferably in arange of about 300 pmol/mL to about 2,000 pmol/mL, and most preferablyin a range of about 400 pmol/mL to about 1,500 pmol/mL. Similar serumlevels of DHSPH are diagnostic of cardiac ischemia.

Although HPLC can be used to detect and quantify cardiac markers,including non-polypeptidic cardiac markers such as SPH in body fluidssuch as serum, other methods of detecting such markers are alsoacceptable. For example, enzymatic assays can be used to indirectlydetect sphingolipids (or other non-polypeptidic cardiac markers) in testsamples. Such assays include, for example, purification of sphingosinekinase from cultured cells which is used in a coupled assay employingpyruvate kinase and its substrate phosphoenblpyruvate to detecthydrolysis. The product of the coupled reaction is pyruvic acid, and thedrop in pH resulting from this product is then detected by a variety ofknown methods such as detecting pH-dependent polymer breakdown thatresults in a measurable change in impedance. Similarly, sphingosinekinase in blood or serum can be detected in a coupled assay employingluciferase to detect ATP hydrolysis. Such assays are suitable forindirectly detecting blood levels of SPH but not S1P or SPC.

Immunodiagnostic assays, using a variety of known methods, can also beused to detect cardiac markers, including non-polypeptidic cardiacmarkers such as sphingolipids and their metabolites, in body fluids,including blood or serum. Antibodies and antibody fragments specific forCer, SPH, DHSPH, S1P, and SPC and other such markers can be produced andused to quantitatively or semi-quantitatively detect the presence of oneor more of such markers in whole blood, serum, or other body fluidsusing standard immunoassays. Similarly, immunoassays that detect thepresence of anti-sphingolipid (or other non-polypeptidic cardiacmarkers) antibodies in body fluids can be used to indirectly test forincreased levels of such marker(s) in patients with chronic conditionsassociated with heart failure, including chronic ischemia and hypoxia.This assay is based on the assumption that patients experiencing suchchronic conditions produce antibodies to these markers as a consequenceof their elevated blood levels by analogy to theanti-lactosylsphingosine antibodies observed in patients with colorectalcancer (Jozwiak W. & J. Koscielak, Eur. J. Cancer Clin. Oncol.18:617-621, 1982) and the anti-galactocerebroside antibodies detected inthe sera of leprosy patients (Vemuri N. et al., Leprosy Rev. 67:95-103,1996).

Detection of one or more secondary markers such as TNFα can be combinedwith detection of one or more non-polypeptidic cardiac marker(s), suchas SPH and/or its metabolites, as an early indicator of heart failure,such as may be caused by cardiac ischemia or hypoxia Because productionof secondary cardiac markers such as TNFα is also associated with heartfailure (such as may be caused by cardiac ischemia) and may induceincreased levels of non-polypeptidic cardiac markers such as SPH and itsmetabolites in body fluids (e.g., blood and serum), the diagnosticcombination of the level of one or more secondary markers such as TNFαand levels of a non-polypeptidic cardiac marker such as a sphingolipidserve as a more sensitive indicator of heart failure. Accordingly, theproduct of the levels of the non-polypeptidic cardiac marker and thesecondary marker(s) can be used to provide a quantitative measure ofrisk of ischemia or hypoxia referred to as the “Myocardial Risk Factor”(MRF).

Detection of non-polypeptidic cardiac markers, such as sphingolipids(including SPH and/or its metabolites) at levels characteristic ofischemia, hypoxia, or other conditions causally related to heartfailure, preferably using a test kit, is useful for identifying theseconditions in angina patients or individuals at risk for ischemic heartdisease. The assay is also useful for diagnosis of AMI and other formsof heart failure. The present invention is useful for simple screeningof persons at risk for heart disease for ischemic or hypoxic conditionsbefore traditional symptoms are detected. The invention is also usefulfor following the progress of therapeutic regimes intended to treatmyocardial ischemia, and thus will have important prognostic value.Methods and compositions of the invention can also be used forpreventing the onset of AMI by allowing the patient or a health careprofessional to use the methods of the invention to detect theconditions that would result in AMI and taking preventive measures, suchas angioplasty.

II. Sphingosine Produced by the Cardiac Cells of Experimental Animalshas Pathophysiological Effects Resembling Heart Failure

Sphingosine (SPH; D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol orsphingenine) is a lipid second messenger that the inventor has found tobe endogenous to cardiac muscle tissue (Dettbarn et al., J. Mol. Cell.Biol. 26:229-242, 1994; Sabbadini et al., Biochem. Biophys. Res. Comm.193:752-758, 1993). Work published by the inventor suggests that SPH hasdramatic effects on the ability of the muscle cells to regulate calcium(Dettbarn et al., 1994; Krown et al., FEBS Letters 376:24-30, 1995;Sabbadini et al., J. Biol. Chem. 267:15475-15484, 1992; Webster et al.,J. Mol. Cell. Cardio. 26:1273-1290, 1994). Low levels of SPH blockcalcium movement whereas very high levels have the opposite effect ofinitiating uncontrolled calcium release and overload (Sabbadini et al.,1992). The acute actions of SPH are specific and the sites of action inthe heart are the sarcoplasmic reticulum calcium release channel(Dettbarn et al., 1994; Sabbadini et al., 1992) and the L-type calciumchannel of the surface membranes (Krown et al., 1995; McDonough et al.,Circ. Res. 75:981-989, 1994). The sphingosine derivative, ceramide, hassimilar actions. Cardiac cell contractility is consequently impaired(Kramer et al., Circ. Res. 68:269-279, 1991; Webster et al., 1994).Thus, SPH is a negative inotropic agent and acts as a calcium channelagonist. The calcium deregulation, negative inotropy, and eventualcalcium overload produced by SPH in experimental animal models resemblesthe pathophysiological changes that the heart experiences duringischemia or other forms of heart failure.

The inventor has demonstrated that chronic treatment of neonatal andadult cardiac cells in culture with physiologically relevant levels ofSPH and its immediate metabolite, S1P, results in the activation ofcardiomyocyte cell death by apoptosis (Krown et al., J. Clin. Invest.98:2854-2865, 1996). Apoptosis is a form of programmed cell death, anddetermines the size of myocardial infarcts (Kajstura et al., Lab.Invest. 74:86-107, 1996). Sphingosine production has been implicated asan early signaling event in apoptotic cell death in a variety of celltypes (Cuvlilier et al., Nature 381:800-803, 1996; Ohta et al., CancerRes. 55:691-697, 1995; Ohta et al., FEBS Letters 355:267-270, 1994).Activation of the sphing myelin signal transduction cascade is a keyearly event in the cytotoxic (apoptotic) effects of TNFα (Zhang andKolesnick, Endo. 136(10):4157-4160, 1995), and the inventor has shownthat TNFα can cause significant apoptosis in cultured rat cardiomyocytesapoptosis (Krown et al., J. Clin. Invest. 98:2854-2865, 1996).

Activity of the enzyme sphingomyelinase (SMase), an enzyme likelyactivated by TNFα in heart tissue (Oral et al., J. Biol. Chem.272:4836-4842, 1997), is increased in the acidotic hearts ofexperimental animals (Franson et al., Am. J. Physiol. 251(5 pt2):H1017-H1023, 1986). SMase is the principle enzyme responsible for SPHproduction in cells and the inventor has localized this enzyme to muscletissue (Sabbadini et al., 1992). There is also evidence from animalmodels of ischemia that the levels of the immediate precursor of SPH,ceramide, are increased in ischernic brain tissue and that ceramidelevels are a consequence of increased sphingomyelin breakdown (Kubota etal., Japan J. Exp. Med. 59:59-64, 1989).

Other supporting data indicate that sphingomyelin levels, the precursorof ceramide and sphingosine, increase in hypoxic experimental animals(Sergeev and Gribanov, Kosm. Biol. Aviakosm. Med. 15:71-74, 1981),although others have found that sphingomyelin levels decrease in thecerebral cortex of ischemic rats commensurate with increased levels ofceramide (Kubota et al., 1996). While not wishing to be bound by aparticular theory, these data support the understanding that theconditions created during hypoxia and ischemia cause the activation ofSMase and the subsequent abnormal elevation of cardiac cell SPH levels.The lysosomal isoform of SMase (acidic or aSMase) could be activated bythe acidic conditions of hypoxia and could complement activation of theplasma membrane isoform of SMase (neutral or nSMase). The nSMase ofcardiomyocytes is likely activated by TNFα. TNFα is released fromischemic cardiac tissue and the TNFα-induced SPH production is an earlyevent in cardiac ischemia.

This invention is in part based on the belief that an early event incardiac ischemia is TNFα-induced sphingolipid production followed bysphingolipid-dependent acidosis that results in additional sphingolipidsynthesis by the acidic form of aSMase, whose source is the lysosome.Sphingosine is a well-known inhibitor of protein kinase C and the systemof Na/H exchange which is activated by the kinase to extrude unwantedacid (Lowe et al., J. Biol. Chem. 265:7188-7194, 1990). As disclosed inSection I, above, cardiac hypoxia, ischemia, and other conditions whichcause heart failure can create a cycle whereby the acidic conditions ofthe ischemic, hypoxic, or otherwise failing heart stimulate excesssphingolipid production, leading to uncontrolled release of calcium fromthe sarcoplasmic reticulum membranes and the L-type calcium channel,thus preventing the cell from regulating its beat-to-beat contractilebehavior.

Deregulated heart calcium levels can also exacerbate the situation bypromoting Na/Ca exchange and indirectly acidifying the cell bystimulation of the Na/H exchanger (Gottlieb et al., Proc. Natl. Acad.Sci., USA 92:5965-68, 1995). Sphingolipid-mediated acidosis andsubsequent calcium deregulation activate the cell death program andresult in apoptosis. In the end, cardiac function suffers from the lossof cells by apoptosis as well as the negative inotropic effects of SPHand pH on surviving cardiomyocytes.

Cell culture studies performed in the inventor's laboratory havedemonstrated that cardiomyocytes can “secrete” SPH into the(cell-conditioned) culture medium (SPH 700 pmol/mL). These observationsshow that SPH and its metabolites could be leaked into the blood fromcardiac cells experiencing the hypoxia and acidosis brought about byischemia. Yatomi et al. reported that S1P is present in human plasma andserum (Yatomi et al., J. Biochem. 121:969-973, 1997). No othersphingolipids, including SPH, were measured, and these workersspeculated that S1P was released from platelets during clotting. Plasmawas incubated with 3H-sphingosine for as long as 2 hours to determine ifS1P could be formed from any component of plasma. The SPH was stable for2 hours in plasma and only platelet-rich plasma converted SPH to S1P,suggesting that the platelets were the source of S1P. Significantly, thesource of SPH for S1P formation by platelets was not discussed, nor wasa potential role of SPH and/or S1P in cardiac ischemia. In contrast, andwhile not wishing to be boundby a particular theory, the presentinvention is based on the understanding that the SPH released fromcardiac cells during the early stages of cardiac ischemia leaks is“secreted” or otherwise escapes into the blood from cells damaged by thehypoxic or ischemic conditions, and is acted upon by sphingosine kinasepresent in blood platelets. The S1P released from the platelets thenstimulates thrombus formation. Thus, the SPH released from cardiac cellsdamaged by hypoxic or ischemic conditions eventually results in theproduction of a myocardial infarction.

III. Tumor Necrosis Factor Alpha (TNFα)

At the molecular level, pro-inflammatory cytokines such as tumornecrosis factor alpha (TNFα) have been implicated in the pathophysiologyof ischemia and hypoxia. Elevated serum TNFα levels occur during hypoxicconditions associated with cardiac ischemia and reperfusion injury, andcirculating TNFα levels are markedly increased after acute myocardialinfarction (Herskowitz A. et al., Am. J. Pathol. 146-419428, 1995;.VaddiK. et al., Circ. 90:694-699, 1994; Lefer A. M. et al., Science 24:61-63,1990; Maury C. P. J. & A.-M. Teppo. J. Intern. Med. 225:333-336, 1989).Reduction in serum TNFα levels is associated with improvements inischemic conditions (Hennein H. A. et al., Circ. 88(4):I-247, 1993). Inhuman patients suffering from chronic heart disease, high crum levels ofTNFα are detectable and increased TNFα levels occur immediately aftercoronary bypass surgery (Levine et al., New Eng. J. Med. 323:236-241,1990; Deng M. C. et al., Eur. J. Cardiol. 9:22-29, 1995; Hennein H. A.et al., J. Thorac. Cardiovasc. Surg. 108:626-35, 1994).

Pro-inflammatory cytokines, such as TNFα, interleukines 1, 2, and 6(IL-1, IL-2, and IL-6), are generally produced by myeloid-derived cellssuch as macrophages, neutrophils and lymphocytes (Kelker H. et al., Int.J. Cancer 36(1):69-73, 1985; Cuturi M. et al., J. Exp. Med.165:1581-1594, 1987; Sung S. et al., J. Clin. Invest. 84(1):236-243,1989; Liebermann A. et al., Proc. Natl. Acad. Sci. USA 86:6348-6352,1989; Lindemanm A. et al., J. Clin. Invest. 83(4):1308-1312, 1989).Smooth muscle and endothelial cells have been suggested as a source ofTNFα (Warner, S., and P. Libby, J. Immunol. 142:100-109, 1989; Libby,P., et al., Am. J. Pathol. 124:179-185, 1986). It has also beenpostulated that the heart is a source of TNFα (Giroir B. et al., J.Clin. Invest. 90:693-698, 1992; Giroir P. B. et al., Am. J. Physiol.267:H118-H124, 1994; Gurevitch J. et al., J. Am. Coll. Cardiol.28(1):247-252, 1996). Ischemic rat hearts perfused in a Langendorffapparatus have been reported to secrete TNFα into the effluent duringthe first minute of reperfusion (Gurevitch et al., 1996).

IV. Heart Cells are the Source of Serum TNFα and SPH

Data collected in connection with the experiments which gave rise tothis invention demonstrate that both neonatal and adult ratcardiomyocytes in culture, devoid of fibroblasts and endothelial cells,are capable of secreting large amounts of TNFα in response to thebacterial endotoxin, lipopolysaccharide (LPS), which is a well-knowsecretagogue for the cytokine. The amount of secreted TNFα can reach1500 pg/mL, which is within the range of TNFα that is capable ofproducing significant apoptotic cell death in cardiomyocytes (Krown etal., 1996). Further supporting the contention that heart cells are asignificant source of TNFα is data showing that TNFα levels in thepulmonary arteries of human subjects undergoing balloon angioplasty isgreater than the serun levels of TNFα found in the femoral veins of thesame patients, which data suggests that, during ischemia (induced byballoon inflation), the ischemic heart tissues produce TNFα which thenis released into the general circulation from the coronary sinuses andthe pulmonary artery. The TNFα in the pulmonary artery of coronaryangioplasty patients correlates well with changes in pulmonary arterySPH levels. Based on these data, it is believed that the cardiac sourceof TNFα is a major stimulus for cardiac cell SPH production.

In sum, the above data indicate that the elevated serum SPH and TNFαseen in various forms of myocardial ischemia, such as occurs duringcoronary angioplasty, results from SPH and TNFα released into thecirculation by ischemic heart cells.

V. Neither SPH Nor TNFα are Elevated in the Serum as a Result ofSkeletal Muscle Ischemia

Since it has previously been demonstrated that SPH is present as asignaling molecule in skeletal muscle (Sabbadini et al., 1993), it wasimportant to determine if skeletal muscle could be the source of serumSPH. Skeletal muscle mass represents 30-40% of total body weight andcould represent a very large source of serum SPH. To confirm that thesource of serum SPH is specifically associated with cardiac ischemia andnot skeletal muscle, several Olympic athletes and Navy subjects weretested for serum SPH before and after inducing severe skeletal muscleischemia. Skeletal muscle ischemia was induced by asking the subjects toexercise to exhaustion on treadmills placed in a 49° C. room, and wasconfirmed by measuring serum lactate. Prior to the exercise regime,serum SPH averaged 5.18±4.5 pmol/rL (n=4) and slightly decreased to alevel of 4.02±3 pmol/mL after exhaustive exercise. Moreover, these serumSPH values were substantially lower than those observed in the ischemicpatients described above. Importantly, serum TNFα levels were notincreased in these subjects undergoing severe skeletal muscle ischemia.For example, serum TNFα values for the military personnel were 1.22±0.49pg/mL before exercise and rose insignificantly to 1.39±0.23 gimL afterexercise for 20 min. at 49° C. (120° F.) ambient temperature.

VI. Determination of the Predetermined Marker Value

In certain embodiments of the present invention, the level of thenon-polypeptidic and/or secondary cardiac marker(s) or the MRFcalculated for a test sample is compared with a predetermined value forthat marker in order to determine if evidence of heart failure, such asmay be induced by cardiac ischemia or hypoxia, exists. The predeterminedvalue for one or more of such markers can be established by one of atleast two ways. For example, it can be established by gathering datafrom the mammal (e.g., a human) at risk of AMI prior to the onset ofsigns for heart disease, or by testing other healthy mammals inpreferably the same species and age group as the patient.

In the first method, the physician treating the patient may determinethat the patient, based on statistical, genetic, familial, or otherfactors generally known in the art of medicine, is at risk of an AMI.The physician can then determine the level of one or morenon-polypeptidic cardiac markers or the MRF for the patient to establisha baseline. Alternatively, or in addition, the physician may alsodetermine the level of one or more secondary markers (e.g., TNFα, IL-1,2, 6, or another cytokine) to establish a baseline. The methods of theinvention provide for the comparison of the level of thenon-polypeptidic cardiac marker(s) and/or secondary marker(s) in thepatient with this baseline in order to detect impending heart failure,such as may be caused by cardiac hypoxia or ischemia.

In the second method, the physician or other health care professional,including a medical statistician, can determine the level of the cardiacmarker(s) or the MRF in individuals determined to be healthy by aphysician. The levels of the individuals in the same age group can begrouped together and their average and standard deviation determined.This value will represent the predetermined value to which the level ofthe cardiac marker in the patient will be compared in order to detectcardiac hypoxia or ischemia.

VII. Early Detection can Lead to Prevention of Acute MyocardialInfarction

The methods and compositions of the present invention allow for earlydetection of cardiac ischemia or hypoxia, i.e., conditions that lead toAMI and other forms of heart failure. Using the instant methods, onceischemia or hypoxia has been detected, the patient can present himselfor herself to an emergency medical facility, where measures can be takenin order to prevent heart failure from occurring. These measuresinclude, but are not limited to, angioplasty, coronary bypass surgery,or administration of one or more anticoagulant or thrombolytic drugs.

The purpose of such preventive measures is to alleviate the ischemic orhypoxic conditions prior to the onset of AMI or other types of heartfailure. In contrast, today the above measures are used after an AMI hasoccurred, or while the patient is experiencing heart failure. Thepresent invention, however, allows for early detection of conditionswhich cause heart failure, prior to the onset of the symptoms and thetissue damage associated therewith.

VIII. Determining Prognosis Following a Cardiac Procedure

The methods and compositions of the present invention allow physiciansand other health care professionals to determine the success of acardiac procedure immediately following the procedure. For instance,following angioplasty or stent placement in a cardiac artery, the levelof one or more cardiac marker(s) or the MRF can be monitored asdescribed herein to determine whether the ischemic or hypoxic conditionsare being alleviated. Thus, the success of the operation can beimmediately determined. If the procedure did not result in the desiredresults, as determined by the level of the cardiac marker(s) or the MRFmeasured, then further procedures can be employed prior to the patientsuffering an complete or partial heart failure.

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

General Procedures

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in theart. Unless mentioned otherwise, the techniques employed or contemplatedherein are standard methodologies well known to those of ordinary skillin the art. The following chemicals, assays and procedures were used toobtain the results presented herein. Those skilled in the art willappreciate that other sources of reagents and well known methods couldbe substituted without departing from the scope of the invention.

Chemicals

Chemicals were obtained as follows: D-Sphingosine[D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol] from Matreya, Inc.(Pleasant Gap, Pa.); sphingosine-1-phosphate from Biomol (PlymouthMeeting, Pa.); o-Pthalaldehyde from ICN Biochemicals (Cleveland, Ohio)and HPLC grade methanol from Fisher Scientific (Tustin, Calif.). Otherchemicals, including sphingosylphosphorylcholine andDL-erythro-dihydrosphingosine, were obtained from Sigma Chemical Co.(St. Louis, Mo.).

Stock solutions of sphingosine and other sphingolipids were prepared ascomplexes with fatty acid-free bovine serum albumin (BSA) to providesolutions of the compounds that are essentially free of micelles ororganic solvents.

Sphingolipid Extraction and Chromatographic Detection Methods

Sphingosine levels were determined by HPLC performed essentially asdescribed previously (Sabbadini, et al., 1993). Briefly, fresh tissuewas excised from the animal and homogenized at 5° C. in the presence offour volumes of 50 mM potassium phosphate buffer, pH 7.0. Similarly, SPHcan be extracted from whole blood or serum samples. Samples (300 μL) ofthe crude homogenates, blood or serum were added to 750 μL of achloroform:methanol solution (1:2) and vortexed several times.Chloroform (about 500 μL) was then added, followed by 500 μL of 1 MNaCl. The extract was centrifuged briefly to promote phase separationand the upper aqueous phase was removed by aspiration. Then, 500 μL of 1M NaCl was added, and the centrifugation and aspiration steps wererepeated. Residual chloroform was removed by vacuum drying for 30 min.in a vacuum centrifuge (SpeedVac, Savant Instruments, Inc., Farmingdale,N.Y.). The residue was suspended in 750 μL of 0.1 M KOH inchloroform:methanol (1:2). The suspension was bath-sonicated andincubated at 37° C. for 1 hr. When the extract had cooled to roomtemperature, 500 μL of chloroform and 500 μL of 1 M NaCl were added, thesample was mixed, centrifuged and the upper phase removed by aspiration.The NaCl extraction step was repeated twice and the organic phase wasvacuum dried with centrifugation for 30 min. The extracts werederivatized for 10 min. with 50 μL of a solution of 0.5 mg/mLo-pthalaldehyde (OPA), 3% boric acid, pH 10.5, and 0.05%β-mercaptoethanol. Then, 50 μL of methanol and 350 μL of HPLC runningbuffer (5 mM phosphate, pH 7.0, in 90% methanol) were added to eachsample and the OPA-derivatized samples were analyzed by HPLC usingstandard methods on a Waters HPLC Maxima 820 Chromatography Workstation(Millipore Corp., Ventura, Calif.), including a Waters 470 scanningfluorescence detector. Fluorescence was detected at an excitationwavelength of 340 nm and an emission wavelength of 455 nm.

The sphingolipids were separated by reverse-phase chromatography on a250 H 7 mm C18, 300 Angstrom pore Brownlee column (Applied Biosystems,Foster City, Calif.) fitted with an Aquapor C18 guard column. Sampleswere run isocratically at 1.25 mL/min using the running buffer,resulting in an efficiency of extraction of about 50% based on therecovery of SPH standards. The retention times for the standards were:about 15 min for SPH; about 5 min for S1P, and about 20 min for SPC.

Example 1

Serum SPH Levels in Human Patients Experiencing Cardiac Ischemia

Serum samples were taken from patients presenting themselves to anemergency medical facility, under a strict human subject protocols.Three patient groups were examined for serum levels of both SPH andTNFα: (1) patients suspected of AMI and subjected to exercise stresstesting; (2) patients undergoing coronary angioplasty; and (3) patientsin the early phases of acute myocardial infarction.

Serum from three control groups not exhibiting any clinical symptoms ofmyocardial ischemic disorders was also tested. These control groupswere: (1) age-matched subjects (47 to 79 yrs old) enrolled in an adultfitness program; (2) healthy military personnel at rest and exercisingto exhaustion on treadmills at 49° C. (120° F.) ambient temperature; and(3) athletes at an Olympic Training Center at rest and exercising toexhaustion on treadmills at 49° C. ambient temperature.

Patients with confirmed myocardial ischemia had significantly higher SPHlevels than any of the control groups. Serum SPH levels for the militarypersonnel and Olympic athletes were combined as one control group (n=6)with resulting serum SPH levels of 4.18±1.8 pmoL/mL. Serum SPH for theage-matched control group (n=15) averaged 99.3±32.4 pmol/mL. When thethree ischemic patient groups were combined as one group, an averageserum SPH level of 697±0.7 pmol/mL was obtained. This value was about7-fold higher than the age-matched control group and about 160-foldhigher than well-conditioned military personnel and athletes undergoingsevere exercise stress.

For comparison with the SPH levels detected, the levels of biochemicalmarkers known to be associated with cardiac tissue damage were alsodetermined. For sixteen of the eighteen ischemic patients, the increasedSPH levels detected were consistent with high levels of CK (in the rangeof 17-810 U/mL) and high levels of CKMB (in the range of 0.62-33.2mg/L). Of the eighteen ischemic patients, two patients (P2, P3) who hadhigh CKMB mass levels did not have abnormally high levels of SPH. Onepatient (P2) had a serum SPH level (120 pmol/mL) that was onlymoderately higher than the average of the age-matched controls, but hadhigh levels of CKMB (33.2 mg/L) and CK (810 U/L), indicative of AMI. Theother patient (P3) displayed a negligible level of serum SPH (18.1pmol/mL), a normal CK level (161 U/L) and a high CKMB level (18.4 mg/L).The moderate-to-low SPH levels detected for these two patients representa 11% false negative rate (2 of 18), because both patients wereconsidered ischemic based on other indicators. The inclusion of the SPHdata for these two patients (P2 and P3) in the AMI group also accountsfor the somewhat lower SPH levels in the AMI group. (587±7 pmol SPH/mL),compared to the SPH levels for the ischemic patients undergoing coronaryangioplasty (885±123 pmol/mL).

Based on the results obtained with the AMI patients tested, a relativelyhigh serum SPH level is an effective early indicator of ischemia. Thiswas further confirmed by the results obtained in the followingindividual studies.

Three Case Histories

Case History No. 1

Patient T2 was a 47 year-old female who presented herself to theemergency room of a hospital with complaints of angina Blood sampleswere drawn and the patient was then referred to a hospital's exercisestress test facility. The patient passed the treadmill test, showing noevidence of cardiac ischemia or AMI as detected by an ECG administratedduring the stress test and by serum enzyme levels analyzed during herperiod of evaluation in the hospital. The patient was discharged fromthe hospital, but returned three weeks later with evidence of an AMI asdetermined by high serum levels of CK and CKMB and other clinicalsymptoms.

Analysis of this patient's serum SPH demonstrated that, at the time ofher first visit to the emergency room, she had a very high level of SPH(810 pmol/mL). When she returned to the hospital with an AMI, her SPHlevel was even higher (greater than 1200 pmol/mL). Thus, the serum SPHlevel was an early indicator of ischemia and more predictive of hercardiac condition than the exercise stress test or the serum CK and CKMBanalysis, both of which showed no evidence of cardiac ischemia or AMI onher first visit to the emergency room.

Case History No. 2

Patient MI-12 was a male who was admitted to the hospital with aconfusing clinical presentation. When admitted, his CK level (126 U/L)was normal, but his CKMB level (3.8 mg/L) was elevated. His serum SPHlevel at admission was also high (732 pmol/mL), indicating ischemia. Atabout five hours after admission, this patient had an AMI. This resultshows that the serum SPH level, but not by the CK parameter, accuratelydetected his ischemic condition. Thus, the results of the SPH test,alone or combined with the CKMB parameter, were predictive of imminentAMI.

Case History No. 3

This patient was a 58 year-old female who was admitted to the hospitalfor an initial evaluation of her heart condition. During the initialevaluation, blood was drawn and the serum showed normal CKMB (0.632mg/L) but moderately high SPH (300 pmol/mL). Four days later shereceived a coronary bypass. After alleviation of the ischemia as aconsequence of the bypass procedure, her serum SPH level wassignificantly reduced to 7.31 pmol/mL, whereas her serum CK (934 U/L)and CKMB (88.9 mg/L) levels remained high. This is consistent with SPHbeing an accurate indicator of ischemia, and the enzymes CK and CKMBbeing indicators of myocardial damage (i.e., large molecular weightcytoplasmic proteins that are released from necrotic myocardial cells).Thus, detection of elevated levels of serum SPH is an early diagnosticof ischemia or hypoxia because it is produced by cardiomyocytes beforesignificant cell necrosis has occurred.

These results show that serum SPH is more predictive of the early stagesof ischemia and imminent AMI than current methods generally used indiagnosis. Moreover, serum SPH is quantitatively related to the earlyevents that precede cardiac cell death, in contrast to other biochemicalmarkers (e.g., CK, CKMB and troponin) that are released after celldeath. Thus, routine screening of patients for serum SPH can aid in theearly diagnosis of coronary artery disease and identification ofpatients at high risk of heart disease who can be treated to preventAMI. Quantitative detection of a single sphingolipid such as SPH,however, may be subject to variability which can be minimized bycombining the measurement with detection of serum TNFα a to provide amore general index of risk of cardiac ischemia or hypoxia.

Example 2

In vitro Sphingosine Production from Cardiac Tissue Under IschemicConditions

To demonstrate that cardiac ischemia results in excess SPH production,tissue levels of SPH were examined in adult rabbit hearts subjected toretrograde coronary perfusion with hypoxic (ie., low oxygen) conditions(95% CO₂; 5% O₂) or with normal Krebs buffers (containing 5% CO₂; 95%O₂). The hearts were removed, quickly homogenized, and sphingolipidswere extracted and detected as described above.

HPLC analysis of the extracts revealed significant increases (20-fold)in tissue SPH levels for hearts perfused with buffer containing 95% CO₂when compared to control conditions. Moreover, these increases occurredafter only 5 min. of treatment.

SPH is a cationic amphipathic lipid that can partition into whole bloodand other body fluids. Therefore, the relative amounts of SPH weredetermined in whole blood and serum obtained from humans, rats, andrabbits. SPH was found predominantly in the serum, the preferred bodyfluid for measuring SPH levels for detecting cardiac ischemia orhypoxia.

In order to determine the time-dependent stability of SPH in serum, ahuman serum sample was obtained and allowed to sit at 22° C. for 5 hrsbefore sphingolipid and HPLC analysis. Serum samples were also spikedwith commercially available SPH and similarly stored at roomtemperature. Neither the aged control serum samples nor the spikedsamples showed appreciable differences in SPH levels compared to samplesthat were assayed immediately after collection or preparation of thespiked samples, indicating that SPH does not undergo degradation ifserum samples were not assayed immediately after collection.

Example 3

Myocardial Risk Factor (MRF)

Serum levels of TNFα also increase in cardiac ischemia and correlatewith the serum SPH levels detected in angioplasty patients. The productof these two parameters (e.g., the levels of a non-polypeptidic cardiacmarker and a secondary [or tertiary] cardiac marker) is referred to as a“myocardial risk factor” (MRF) and is a useful quantitative indicator ofan individual's possibility of injury resulting from myocardialischemia. Because MRF can be calculated using different cardiac markers,it is important that the markers used in a particular MRF be specified.

Here, TNFα was measured using standard methods in an enhanced ELISAdouble antibody capture assay (using a Quantikine HS kit (Cat No.HSTA50) kit, R+D Systems, Minneapolis, Minn.). Human serum samples (200μL) were assayed and compared to recombinant human TNFα standards usingthe assay procedures provided by the manufacturer.

Serum TNFα and SPH levels were determined from samples taken from thepulmonary artery of a patient (A5) experiencing the periodic ischemia(e.g., during balloon inflation) and reperfusion (e.g., when the arteryis cleared) that occurs during successful angioplasty. Serum TNFα andSPH levels were both elevated prior to initiation of the angioplastyprocedure (from =20 min to 0 min), Neither the TNFα nor the SPHincreased during the ischemic period. They were already high and wentdown after reperfusion. Both TNFα and SPH parameters showed a biphasicresponse, with initial decrease after angioplasty followed by a steadyrise back to pre-angioplasty levels. The time courses were similar withTNFα levels falling slightly ahead of SPH in time. This is consistentwith TNFα being the trigger for SPH production.

Similarly, serum TNFα levels were assessed for 19 patients undergoingcoronary angioplasty and for seven AMI patients. For comparison, serumTNFα levels were determined for two control groups (age-matched controlsand healthy military personnel). Serun TNFα levels were highest in theAMI patients (5.2±0.6 pg/mL), and somewhat lower in the pulmonary arteryblood taken from the angioplasty group before the procedure was done(3.6±0.74 pg/mL). The patients had significantly higher serum TNFαlevels (4.04±0.57 pg/mL) compared to those of the age-matched controlgroup (“Controls”) of 2.43±0.32 pg/mL, and the healthy military subjects(“Athletes”) of 1.22±0.29 pg/mL.

By comparing the results, it can be seen that serum TNFα levels showedthe same trends as seen for the serum SPH levels in the three groups'tests. That is, the athletes had the lowest levels of SPH and TNFα, theage-matched controls had higher levels of SPH and TNFα, and the patientshad the highest levels of SPH and TNFα. Therefore, a more accuratemeasure of on-going myocardial ischemia can be obtained by combining twoor more parameters, e.g., the levels of a non-polypeptidic and asecondary or tertiary cardiac marker trending in the same direction, tocalculate a MRF. When SPH is the non-polypeptidic cardiac marker andTNFα is the secondary cardiac marker, the MRF is the product obtained bymultiplying the numerical value of the serum TNFα level and thenumerical value of the serum SPH level.

Representative MRF data (using SPH and TNFα levels) for all ischemicpatient groups in this experiment were combined and compared toage-matched and healthy military controls. The MRF of the ischemicpatients (2820) was about 12-fold higher than that of the age-matchedcontrol group (238) and about 440-fold higher than that of the healthymilitary group (6.3). Thus, the MRF value distinguished ischemicpatients from controls to a greater extent than did either serum SPH orserum TNFα measurement alone. Moreover, the calculated MRF value reducedthe occurrence of false negatives for either parameter alone.

Table 1, below, shows the SPH/TNFα MRF value calculated for sevenpatients. The values were calculated by multiplying the SPH value(approximated to the nearest whole integer) and the TNFα value(approximated to the nearest one-tenth) to produce the MRF value (to thenearest whole integer). Six of the seven angioplasty patients had a MRFin the range of about 1,800 to about 3,000 range. The mean MRF value forthese six patients was 2,326. Only one patient had a MRF outside of thatrange (patient A1 with MRF of about 16,000). The mean MRF for all sevenpatients is shown in Table 1. Based on the MRF values of the majority ofthe patients for whom the calculation was made, patient A1 appeared tohave an atypically high level of ischemia, with the TNFα level beingsignificantly higher than that of the other six patients. If the SPHlevel of patient A1 is multiplied by the mean TNFα value of the othersix patients (2.9), the MRF value would be 3,254 which is close to therange seen for the other six patients.

TABLE 1 SPH TNFα CK Patient (pmol/mL) (pg/mL) MRF (U/mL) A1 1,122 14.416,157 175 A4 868 2.9 2,500 110 A5 1,000 2.7 2,700 50 A6 582 3.2 1,84533 A8 772 3.1 2,393 N/A A10 1,404 1.9 2,668 41 A11 447 4.1 1,833 54Mean: 8851 4.6 4,302 77 S.D.: 325 4.4 5,240 55 S.E.: 133 1.8 2,139 25

There were no false negatives for the SPH value detected in theangioplasty group; showing that an elevated serum SPH level is anaccurate predictor of ischemia Serum SPH was predictive of the earlystages of ischemia and imminent AMI more frequently than other currentlyused methods, although false negative results were detected with a fewAMI patients. Moreover, the level of SPH detected provides aquantitative measurement of early events that precede cardiac celldeath. In contrast, other biochemical markers (e.g., CK and CKMB) appearto be more indicative of later events subsequent to cardiac cell death.Because measurement of serum SPH provides information on the level ofcardiac ischemia even in the absence of other clinical indicators, theassay is also useful for monitoring the efficacy of cardiac treatments(e.g., bypass surgery or angioplasty). Testing of serum SPH levels isuseful as a routine diagnostic of cardiac ischemia and hypoxia,permitting patients at risk of heart disease to be identified andtreated. The method is also useful for monitoring patients during orafter treatment for cardiac conditions to detect the level of cardiacischenia or hypoxia as an indicator of the success of the treatment.

Although the results used to calculate the MRF presented above werebased on HPLC-detection of SPH and ELISA detection of TNFα, it will beappreciated by those skilled in the art that a variety of assays can beused to detect any of the diagnostic sphingolipids and TNFα. Preferably,a kit that includes assays for TNFα and sphingolipids is used to providea measure of the MRF. For example, anti-TNFα and anti-SPH antibodies/oran enzyme assay for sphingolipids are combined in a kit to assess theMRF value. For example, known biosensor technology can be used for thedetermination of two or more analytes in blood or serum. An algorithmmay be used in the kit to calculate the MRF value, which may beparticularly advantageous for detecting all three importantsphingolipids, SPH, S1P and SPC, as well as TNFα, and determining aseries of M values for all of the combinations of sphingolipid and TNFα,or for determining a single MRF value that is the product of all themeasured sphingolipid levels and the TNFα level.

Example 4

TNFα Associated with Ischemia is Produced by Heart Cells

To demonstrate the myocardial cell origin of TNFα, both neonatal andadult rat cardiomyocytes in culture, devoid of fibroblasts andendothelial cells, were tested for production of TNFα.

Neonatal ventricular myocytes were dissociated from hearts obtained fromone to four day old Sprague-Dawley rats essentially as describedpreviously (Shields et al., J. Biol. Chem. 263:12619-12618, 1988).Ventricles were finely minced and dissociated with 0.5 g/L trypsin, 0.2g/L EDTA in a calcium-free and magnesium-free Hank's buffered saltsolution (Sigma, St. Louis, Mo.). The tissues were agitated, pelletedgently by centrifugation and the trypsin digestions were repeated fivetimes. The supernatants containing cells in suspension were combinedwith DMEM/F 12 medium, filtered through 125 μm nylon mesh, and thefiltrate was centrifuged. The pelleted cells were resuspended inDMEM/F12 medium and plated on fibronectin-coated glass coverslips inDMEM/F12 medium plus 10% fetal calf serum (FCS).

Freshly dissociated adult ventricular myocytes were prepared from heartsof adult (200 to 350 g) Sprague-Dawley rats by enzymatic dissociationusing a Langendorff retrograde aortic perfusion apparatus. Afterperfusing the hearts with collagenase (Type II, WorthingtonBiochemicals, Freehold, N.J.), rat ventricles were diced and incubatedfor 30 min at 37° C. in 10 to 15 mL of 0.58 mg/mL collagenase inoxygenated Tyrode's solution (140 mM NaCl, 5.4 mM KCl, 5.0 mM MgCl₂, 1.0mM CaCl₂, 10 mM HEPES, 0.25 mM NaH₂PO₄, pH 7.3). The dissociatedmyocytes were plated on laminin-coated (50 μg/mL) culture dishes andcultured for 18 hr in DMEM/F12 plus 10% FCS, in the presence or absenceof the test agent (e.g., LPS or control buffer).

The results showed these cells were capable of secreting high amounts ofTNFα in response to the bacterial endotoxin, LPS, which is a well-knownsecretagogue for the cytokine. The amount of secreted TNFα can reachabout 1500 pg/mL which is within the range of TNFα capable of producingsignificant apoptotic cell death in cardiomyocytes. Additionally, invitro experiments with cultured rat cardiomyocytes stimulated by LPSshow that a significant amount of SPH (700 pmol/mL) is, also secretedfrom the cells. These results demonstrate that heart cells are a sourceof secreted SPH as well as TNFα. Further, these in vitro results areconsistent with the in vivo results in which TNFα and SPH levels in thepulmonary arteries of human subjects undergoing balloon angioplasty weregreater than the serum levels of TNFα found in the femoral veins of thesame patients. The data indicate that the TNFα levels in the pulmonaryartery of coronary angioplasty patients correlated well with changes inpulmonary artery SPH levels during the same treatment. Both the in vitroand in vivo data indicate that the elevated serum TNFα and SPHlevels'seen in myocardial ischemia (e.g., during coronary angioplasty)likely results from TNFα released by ischemic heart cells into thecirculation.

The elevated serum SPH levels detected in the ischemic patients was notproduced by skeletal muscle ischemia as demonstrated by assaying serumsamples from healthy athletes and military personnel before and afterinducing severe skeletal muscle ischemia. The subjects exercised toexhaustion on treadmills placed in a room having an ambient temperatureof 49° C. to induce skeletal muscle ischemia which was confirmed bymeasuring serum lactate. Prior to the exercise regime, serum SPHaveraged 4.18±4.5 pmol/mL, which slightly decreased to 4.02±3 pmol/mLafter exercise. These serum SPH values were substantially lower thanthose detected in ischemic patients. Importantly, serum TNFα levels werenot increased in these subjects undergoing severe skeletal muscleischemia (for example, serum TNFα values for the military personnel were1.22±0.49 pg/mL before exercise and rose insignificantly to 1.39±0.23pg/mL after exercise).

Example 5

Use of Anti-sphingolipid Antibodies to Detect Sphingolipids in WholeBlood or Serum

Anti-sphingolipid (or other non-polypeptidic cardiac markers) monoclonalantibodies (mAb), e.g., mAbs reactive against SPH, S1P, SPC, and DHSPH,are prepared by methods similar to those used in the preparation ofanti-phospholipid antibodies. Briefly, one method of mAb productioninvolves direct immunization of sphingolipid-coated, acid-treatedSalmonella. Minnesota directly into a mouse spleen using known methodsused to make anti-phospholipid mAbs (Reza et al., FEBS Lett.339:229-233, 1994; Umeda et al., J. Immunol. 143:2273-2279, 1989). Forproduction of anti-SPH antibodies, the acid-treated S. Minnesota iscoated with the desired sphingolipid, e.g., SPH, and injected into themouse spleen prior to cell fusion to produce a hybridoma that secretesanti-SPH mAb. Similar methods are used to produce anti-S1P mAb andanti-SPC mAb. Alternately, fatty acid free BSA-sphingolipid conjugatescan be used as the immunogen in order to present unique epitopes to theanimal. Care must be taken to ensure that mAbs are not produced tooxidized lipid or protein-lipid adducts. [see discussion by Witztum etal. in refs. (Horkko et al., J. Clin. Invest. 98:815-825, 1996; Palinskiet al., J. Clin. Invest. 98:800-814, 1996)].

The mAbs are initially used to detect the specific ligand in any of avariety of standard immunoassays, such as, for example, an enzyme-linkedimmunosorbent assay (ELISA), a radioimmuno assay (RIA), by directlabeling of the mAb with a calorimetric label (e.g., colloidal gold orlatex beads), or by indirect labeling of the mAb such as in a sandwichimmunoassay. All of these assays are well known in the art and can bepracticed by the skilled artisan with minimal routine testing todetermine optimal conditions for detecting the specific ligand(s).Preferably, the immunoassay would employ the standard lateralflow-through format or biosensor technology.

Lateral flow formats involve double capture antibody technology wherethe analyte in the blood sample is captured by the first antibodytethered to the substrate. A strept-avidin system is then used to detectbinding of the second antibody.

Biosensor technology typically uses surface plasmon resonance to detectrefractive index changes on the surface of a gold/glass matrix as theantigen (e.g., the sphingolipid) binds to the tethered antibody (e.g.,anti-SPH) [see for example, Kuziemko et al., Biochem. 35:6375-6384,1996, where cholera toxin binding to gangliosides, including lactosylceramides, was studied]. Biosensors are preferred because they are veryrapid (develop in minutes), quantitative, and are amenable to use withmultiple ligands. Algorithms and digital readouts are possible withbiosensors. Biosensor format-based immunoassays for detection of SPH,S1P, and SPC can be performed individually, to provide independentmeasurements of each of the sphingolipids as indicators of ischemiccardiac conditions. Alternatively, a single assay could include multiplemAbs to provide a single measurement of any combination of sphingolipids(e.g., SPH and S1P; SPH and SPC; S1P and SPC; or SPH, S1P and SPC),alone or in combination with one or more other markers, e.g., TNFα.

Example 6

Enzymatic Assay for Serum or Whole Blood Sphingosine

This method involves purification of sphingosine kinase and its use in acoupled assay employing pyruvate kinase and its substratephosphoenopyruvate to detect ATP hydrolysis. The product of the coupledreaction is pyruvic acid and the resulting change in pH is used in a kitthat takes advantage of pH-dependent polymer breakdown technology (asdescribed in Serres, A. et al., Pharmaceutical Res. 13(2):196-201, 1996)and the subsequent changes in impedance that are measured. Sphingosinekinase is isolated from Swiss 3T3 cells or other cells as previouslydescribed (Olivera A., et al., Anal. Biochem. 223(2):306-312, 1994).

The assay includes the following features: The substrate of the teststrip is coated with a pH-sensitive linear terpolymer (e.g., aderivative of poly(N-isopropylacrylamide-co-burylmethacrylate-co-acrylicacid). A blood or serum sample is dropped onto the test strip and thecoupled reaction precedes. As the pH drops by the coupled enzyme assay(the decrease in pH is proportional to the amount of SPH in blood), thepolymer breaks down and exposes the conductor on the test strip. Animpedance measurement is then made which is proportional to the amountof SPH in the blood or serum sample that was dropped onto the strip.

Example 7

Phage Display Assay for Sphingolipid Receptor Isolation

This technique is used to screen phage which encode receptors which bindwith high affinity to the markers, particularly the non-polypeptidiccardiac markers, of the invention, and express them on their surfaces.See U.S. Pat. Nos. 5,223,409 and 5,403,484 for a detailed description ofphage display technology. Bacteria, which express the receptor, are thencloned and used in a biosensor-based kit. This technique can be used todetect the markers used in the practice of this invention, including allimportant sphingolipids, including SPH, S1P, DHSPH, and SPC. Because thetechniques used to isolate receptors for any marker are substantiallythe same, the techniques are described generically herein. This methodis described in more detail in McGregor, D., Mol. Biotech. 6(2):155-162,1996.

Typically, a cDNA library is provided (e.g., a cDNA library that isavailable from a number of commercial sources, including Clontech, SanDiego, Calif.) and then used to create fusion proteins with a membraneprotein of a filamentous male phage such as M13. Phage clones expressingthe fusion protein containing the receptor for the desired marker, e.g.,SPH are detected and isolated using standard ligand binding assays. Thegene for the receptor is then excised from positive clones usingstandard endonuclease restriction enzymes and cloning methods (see,e.g., J. Sambrook, Molecular Cloning, A Laboratory Manual, 2nd Ed., CSHLab. Press, 1989). The gene may also be expressed in a bacterial systemusing standard methods to yield unlimited quantities of the receptor.The receptor is purified using standard methods and is then used todetect the marker for which the receptor demonstrates specificity. Forexample, the purified receptor is tethered to an ELISA plate, to aBiacore dextran surface, to a test strip of any of a number of detectionkits, to biosensor detectors and the like, and used to measure thequantity of a sphingolipid in the blood or serum. Other applicationstaking advantage of marker binding to its membrane receptor are alsoenvisioned.

Example 8

Measurement of Anti-sphingosine Antibodies in Human Blood

This method is based on the assumption that patients experiencingischemia produce anti-sphingolipid antibodies as a consequence ofelevated blood levels of sphingolipids such as sphingosine. It is alsobased on the findings that anti-lactosylsphingosine antibodies have beenobserved in patients with colorectal cancer (Jozwiak W. and J.Koscielak, 1982) and anti-galactocerebrosides were detected in the seraof leprosy patients (Vemuri N. et al., 1996).

The potential antigenicity of sphingosine and its metabolites issuggested by their structures as a cationic amphiphiles (e.g., seeFIG. 1) and by the finding that antibodies can be generated againstphospholipids (Umeda M. et al., 1989) and glycosphingolipids (Vemuri N.et al., 1996). For example, anti-glycosphingolipid antibodies weredetected in the serum of calves experimentally infected with T. saginata(Baumeister S. et al., Parasitol. Res. 81:18-25, 1995). This techniquecan be applied to detecting any sphingolipid or sphingolipid metabolite,including SPH, DHSPH, S1P and SPC, as well as other non-polypeptidiccardiac markers used in the practice of this invention.

To isolate antibodies against non-polypeptidic cardiac markers, e.g.,SPH, from the serum of ischemic patients, one can employ affinitypurification of the antibodies from the serum using a matrix, such asSepharose, to which the marker to be detected is conjugated. Theseantibodies form the basis of an immunological test, using any of avariety of well-known immunological screening methods, that would beeasy to administer and inexpensive to perform for large patientscreenings.

Example 9

Home Monitoring of Cardiac Markers

Individuals who wish to monitor their cardiac marker levels without theaid of a health care professional may use a home monitoring device.Finger stab dipstick technology is widely used for blood glucosemonitoring and this method can be adapted to the measurement ofblood-borne cardiac markers (e.g., non-polypeptidic, secondary, andtertiary markers) in a drop of blood, given the disclosure herein. Thecardiac markers used in the practice of the invention, includingsphingosine and its derivatives, can also be measured in many tissuesand body fluids besides blood, including saliva, sweat, and urine. Otherhome monitoring devices useful in the practice of this invention includethose analogous to in-home pregnancy tests, where the level of aparticular marker or set of markers is measured in urine.

A portable electronic measurement device can also be applied in thepractice of this invention. For instance, a wrist-worn device similar insize and shape to a wristwatch has been developed for monitoring bloodsugar levels in diabetics. For example, the Cygnus GlucoWatch™ platformallows an individual to continuously monitor glucose through intact skinfor more accurate assessment of the analyte at all times and without thediscomfort of the finger stab technique. Similarly, real-time continuousor periodic monitoring of one or more cardiac markers in accordance withthe instant methods could be accomplished by wristwatch platforms. Insuch embodiments, the measurement of non-polypeptidic cardiac markers(and secondary and tertiary markers, if desired) according to theinvention would occur directly through the skin, and would be comparedwith marker levels indicative of cardiac conditions associated withischemia, hypoxia, and others correlating with various forms of heartfailure. Downloading of stored data from such a device having datalogging capability is also envisioned, and would provide the clinicianwith a record of the patient's recent history of marker level changes.

Furthermore, such a device, or other home monitoring device designed tomonitor cardiac marker levels according to the invention, can be used inconjunction with an alarm system that is activated when one or morecardiac marker level (or a calculated index such as MRF) exceeds acertain threshold. The alarm may inform the patient, i.e., wearer, or athird party, e g., a friend or relative,, a health care professional, oremergency response personnel, such as the police, paramedics, or thefire department of a change in the level of the marker(s) beingmonitored. Such an alarm, when transmitted, may also include telemetry.

A home monitoring device according to the invention will preferably beaccompanied by instructions for use. The device may also be accompaniedwith a notice in form prescribed by a governmental agency regulating themanufacture, use, or sale of medical devices, which notice is reflectiveof approval by the agency of the form of the device for home use. Suchnotice, for example, may be the labeling approved by the U.S. Food andDrug Administration, or the equivalent governmental agency in othercountries, for medical devices, or the approved product insert.

Example 10

Sphingolipids as Inhibitors of Protein Kinase C

This embodiment of the invention employs a coupled assay to assesssphingosine levels in blood or other tissues or bodily fluids by takingadvantage of the ability of sphingosine and other lysosphingolipids toinhibit protein kinase C (PKC) (Hannun and Bell, Science 234:670-674,1987; Hannun et al., J. Biol. Chem. 261:12604-12609, 1986). The amountof sphingolipid in the blood sample is quantitatively related to thechange in absorbance at 340 nm as a stoichiometric amount of NADH isoxidized by the coupling system as described by Sabbadini and Okamoto(Sabbadini and Okamoto, Arch. Biochem. Biophys. 223:107-119, 1983).

It has been shown that sphingosine inhibits PKC by preventing DAGbinding to the enzyme (Faucher et al., J. Biol. Chem. 263:5319-5327,1988). Thus, sphingosine may bind directly to PKC via the DAG bindingsite. The sequence for PKCα and its consensus DAG binding site is known(Hurley et al., Protein Science (6):477-80, 1997). Since SPH can alsobind to putative sites on sphingosine kinase and other proteins withwhich is specifically interacts, it quite likely that several proteinshave specific SPH binding sites. Accordingly, the putative sphingplipidbinding site can be cloned using standard techniques, after thescreening of phage display libraries (see above) for colonies, whichexpress the sphingolipid recognition site. Expression cloning of thecDNA of this protein would produce a reagent that could be used in astandard ELISA to detect sphingolipid changes in a blood sample.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themethods, procedures, treatments, devices, and compositions describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Upon reading this specification, changes therein and otheruses will occur to those skilled in the art, each of which isencompassed within the spirit of the invention as defined by theattached claims.

All patents and publications referred to above are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, or limitation or limitations,which is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof,” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.Other embodiments are within the following claims.

I claim:
 1. A kit for detecting a condition which causes heart diseasein a mammal, the kit comprising a composition for detecting a level ofat least one non-polypeptidic cardiac marker in a test sample obtainedfrom said mammal, wherein said kit is able to determine if said level ofsaid non-polypeptidic cardiac marker in said test sample correlates withsaid condition that causes heart disease in a mammal wherein said markeris selected from the group consisting of sphingolipid, sphingosine,sphingosine-1-phosphate, dihydrosphingosine andsphingosylphosphorylcholine.
 2. A kit according to claim 1 wherein saidlevel of at least one non-polypeptidic cardiac marker is measuredquantitatively.
 3. A kit according to claim 1 wherein said compositioncomprises a substrate.
 4. A kit according to claim 3 wherein saidsubstrate is an antibody which binds specifically to a non-polypeptidiccardiac marker.
 5. A kit according to claim 3 wherein said substrate isan antibody which binds specifically to a sphingolipid.
 6. A kitaccording to claim 1 wherein said kit comprises one or more standardshaving a predetermined level of a non-polypeptidic cardiac marker.
 7. Akit according to claim 6, wherein said kit comprises two or morestandards, wherein at least two of said standards have different levelsof said non-polypeptidic cardiac marker.
 8. A kit according to claim 7wherein said kit comprises instructions for comparing the level of saidnon-polypeptidic cardiac marker in said test sample to the level of atleast one non-polypeptidic cardiac marker in said two or more standards.9. A kit according to claim 1 wherein said kit comprises a compositionor device for obtaining a test sample using an invasive method.
 10. Thekit according to claim 1 further comprising a composition for measuringthe level of a second marker.
 11. The kit according to claim 10 whereinsaid second marker is a pro-inflammatory cytokine.
 12. The kit accordingto claim 11 wherein said pro-inflammatory cytokine is TNFα.
 13. A methodof using the kit of claim 1 to detect a condition which causes heartdisease in a mammal.
 14. The kit of claim 1 wherein said heart diseaseis selected from the group consisting of heart failure, cardiac ischemiaand cardiac hypoxia.
 15. The kit of claim 14 wherein said heart failureis selected from the group consisting of acute myocardial infarction,myocarditis, a cardiomyopathy, congestive heart failure and idiopathicheart failure.
 16. The kit of claim 1 wherein said mammal is a human.